Impairment in novelty-promoted memory via behavioral tagging and capture before apparent memory loss in a knock-in model of Alzheimer’s disease

Alzheimer’s disease (AD) is associated with cognitive impairments and age-dependent memory deficits which have been studied using genetic models of AD. Whether the processes for modulating memory persistence are more vulnerable to the influence of amyloid pathology than the encoding and consolidation of the memory remains unclear. Here, we investigated whether early amyloid pathology would affect peri-learning novelty in promoting memory, through a process called behavioral tagging and capture (BTC). AppNL-G-F/NL-G-F mice and wild-type littermates were trained in an appetitive delayed matching-to-place (ADMP) task which allows for the assessment of peri-learning novelty in facilitating memory. The results show that novelty enabled intermediate-term memory in wild-type mice, but not in AppNL-G-F/NL-G-F mice in adulthood. This effect preceded spatial memory impairment in the ADMP task seen in middle age. Other memory tests in the Barnes maze, Y-maze, novel object or location recognition tasks remained intact. Together, memory modulation through BTC is impaired before apparent deficits in learning and memory. Relevant biological mechanisms underlying BTC and the implication in AD are discussed.

Behavioral tasks and procedures. The ADMP task. The ADMP task is an everyday spatial memory task that encourages the animals to encode a unique rewarded location on the day and update it across days. It was composed of habituation, initial training sessions, probe tests to assess memory, and interleaving training sessions 21 .
Habituation. To habituate the mice to dig for rewards, sandwells were placed in the home cage with pellets becoming gradually deeper in the sand over 4 sessions, then they were habituated to digging the sandwell in the centre of the arena. Mice were encouraged to return to the start box to eat the reward. Once 2 rewards were retrieved in less than 5 min, the mice could move on to the regular training schedule.
Training. Each training session consisted of 2 encoding trials and 1 retrieval trial per day separated by intervals of approximately 10 min each. In an encoding trial, one rewarded sandwell was placed in the arena. The mouse was then placed in the start box, consumed a small cue pellet, and entered the arena to search for the sandwell. Mice would return to the start box to consume the pellet before returning to the arena to retrieve the second pellet. The location of the rewarded sandwell was counterbalanced across days and genotypes. During the retrieval trial, one rewarded sandwell was placed at the location that matched to the encoding one and 4 unrewarded sandwells were also added in the arena. The trial was ended when the mouse collected both rewards. The latency (in seconds) to dig at the correct sandwell and the number of errors in digging at the unrewarded sandwells (max 4, min 0, chance 2) were recorded as indices of learning.
Memory probes. After the mice went through initial training, memory was assessed via post-encoding probe tests that consisted of 5 unrewarded sandwells. A 60 s timer was started following the first dig at any sandwell. Time spent at digging the correct (i.e. matching to the encoding location) and incorrect sandwells was recorded. The percentage of correct digging time over total digging time served as the index of memory and chance level was 20%. After the 60 s, the experimenter placed 2 rewards in the correct well to prevent extinction of digging. At least 1 training session interleaved probe tests to prevent extinction of the matching-to-place rule. STM was assessed in a probe test 10-min after 2 encoding trials of 2 pellets per trial. LTM was assessed in a probe test 24 h after 2 encoding trials with 4 pellets per trial (strong encoding) or after 1 encoding trial with 2 pellets (weak encoding). Intermediate-term memory (ITM) was assessed 6 h after 1 encoding trial with 2 pellets. Exploration in a novel box or with novel objects for 5 min may occurred 40 min after encoding to determine if peri-learning novelty improves memory persistence.
Open-field exploration. Mice were placed at the centre of the Perspex box for 5 min. Exploration was recorded via an overhead camera using Anymaze software (Stoelting Co., IL, USA). Time spent in the zones representing the inner 50% and outer 50% area of the box and the distance travelled (not including rearing) were measured. This 5-min trial also served as the first session of six habituations to the Perspex box for the novel object recognition and novel object location tasks. The novel object recognition task. In the sampling phase, mice were given 10 min to explore 2 identical objects placed in opposite corners, 10 cm away from the Plexiglas box walls. In the 5-min test phase, 24 h after the sampling phase, one object had been replaced by a different object. The location of sampling and testing objects, and identity of objects pairs were counterbalanced across animals. Exploration of both objects was scored manually by the experimenter when the animals' head pointed toward and came within 2 cm of the object 36,37 . A discrimination index was calculated using the following formula: novel object exploration (s) − familiar object exploration (s) total object exploration (s) × 100 www.nature.com/scientificreports/ The novel object location task. In the sampling phase, the mice were given 10 min to explore two identical objects placed 10 cm away from the corners, against the same side of the box. In the 5-min test phase, 6 h after the sampling phase, one object was moved to the centre of the opposite wall and the animal was given 5 min to freely explore the new setup. A 6 h delay was used due to unreliable 24-h NOL memory using this sampling duration in a pilot study. Exploration of both objects was scored manually by the experimenter with the criteria describe above. The location of sampling and displaced objects was counterbalanced across animals. A discrimination index was calculated using the following formula: The Y-maze task. The Y-maze task was carried out to assess the animals' spontaneous tendency to alternate among the 3 arms of the maze, often linked to working memory 38,39 . Each mouse was placed at the centre of the maze with the facing arms counterbalanced between mice. The sequence of arm visits was recorded for 5 min. Alternation was defined as the mouse entering 3 different arms consecutively (chance: 22.2%). An alternation index (%) was calculated by using the formula 39,40 : The Barnes maze task. The mice were habituated to the start cylinder, the maze itself, and the escape box for 2 days prior to training. For each training trial, the mice were placed in the start cylinder at the centre of the maze for 10 s followed by exposure to the whole maze with loud noise and bright light. The trial ended when the mouse entered the escape box and was capped at 3 min when the experimenter guided the mouse to enter the escape box. Training consisted of 5 daily sessions of 2 trials in each session. The escape location was kept consistent per animal and counterbalanced across animals. In the STM and LTM probes, 1 and 24 h after training respectively, the mice had 60 s in the maze with white noise and bright light but without the escape box. Time spent in each quadrant was recorded and scored using Anymaze. Mean speed was scored by Anymaze as total distance travelled (m)/total time (sec). The escape box was replaced at the end of the probe to prevent extinction.
Histology. Animals were anaesthetised deeply with pentobarbital and transcardially perfused with 1 × phosphate buffered saline (PBS) then 4% paraformaldehyde around 14 months old. Coronal brains sections (40 µm) were collected. To stain for β-Amyloid, sections were blocked in 10% normal goat serum with 0.1% Triton™ X-100 (Sigma Aldrich) in PBS for 1 h followed by an overnight incubation in primary antibody (1:500 mouse anti-β-Amyloid, 6E10, BioLegend). Sections were incubated in the secondary antibody for 2 h (1:100 antimouse Biotin, Sigma Aldrich), followed by the Avidin-Biotin complex (ABC elite, standard, Vectastain), and 3.3′-diaminobenzine (DAB, Vector Laboratories). Sections were mounted on glass slides and coverslipped with dibutylphthalate polystyrene xylene (DPX, Sigma Aldrich). Note, sections from one App NL-G-F/NL-G-F mouse were excluded due to hippocampal tissue damage in processing. Hippocampal images were acquired via Zeiss, Axio Scan.Z1 (200 micro-sec, 1.72 µm depth of focus). Images were analysed using ImageJ (1.52n) to measure the percentage area with positive 6E10 signal and manual plaque number counting.
Statistics. All statistical analysis was done using SPSS Statistics 24 (IBM). Two-way mixed ANOVAs with sphericity checks were used for reporting 2 factors and interactions, such as training and genotype effects in training errors and latencies. Genotype comparisons in designs with a single factor were run using independent samples t tests and checked for equal variance. If the data did not fulfil normality, a Mann-Whitney U test was run. Comparisons against a fixed value (e.g. chance or 0) were checked for normality and then analysed by 1-sample t tests or a related-samples Wilcoxon signed rank test if normality failed.

Results
App NL-G-F/NL-G-F , compared to wildtype, mice do not display learning or memory impairments in the ADMP task. A series of behavioral tasks were performed, starting with the ADMP task ( Fig. 1a,b). Acquisition of the ADMP task in adulthood was first assessed by the number of errors made during the retrieval trial (Fig. 1c). It was averaged over 7 blocks of 3 training sessions per block and a 2-way ANOVA was performed to assess the training effect, genotype effect and the interaction of the two (Fig. 1c). Both genotypes made a similar number of errors during training (F 1,23 = 0.18, p = 0.7). The effect of training block and the interaction were both insignificant (F 6,138 = 0.76, p = 0.6 and F 6,138 = 0.65, p = 0.7, respectively). Importantly, the errors made during the 7 blocks of training were significantly below chance (block 1 to block 7: t 24 = − 2.71 to − 5.51, p < 0.02 to p < 0.0001), indicating the mice learnt the task early on and persistently performed well. The latency to find the reward during the retrieval trials revealed a highly significant training effect (Fig. 1d, F 2.9,66.9 = 6.45, p = 0.001) and the linear trend was also significant (F 1,23 = 14.94, p = 0.001). This suggests that mice improved in task efficiency over time. The genotype effect and interaction were both insignificant (F 1,23 = 0.76, p = 0.4 and F 2.9,66.9 = 0.56, p = 0.6), suggesting a similar rate of improvement in both groups.
In the STM test, mice showed a significant preference for digging in the previously rewarded (i.e. correct) sandwell compared to chance (Fig. 1e, WT: t 11 = 2.97, p = 0.013, and App NL-G-F/NL-G-F : t 12 = 2.60, p = 0.023), with no difference between the two genotypes (t 23 = 0.15, p = 0.9). In the LTM test, mice showed a significant preference for The novelty-promoted memory comprised of a weak encoding, followed 40 min later by exploration in a box with novel substrates. LTM and ITM were not significantly above chance in both groups. (h-l) The novelty-promoted memory comprised of a weak encoding, followed 40 min later by exploration in a box with novel objects. The percentage of time digging at the correct location was significantly above chance for WT, but not App NL-G-F/NL-G-F mice after exploration of novel objects (k) and in replication (l). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01. www.nature.com/scientificreports/ digging in the correct sandwell (Fig. 1f, WT: t 11 = 4.10, p = 0.002, App NL-G-F/NL-G-F : t 12 = 4.06, p = 0.002). There was no significant effect of genotype (Mann-Whitney U = 53, p = 0.2). Together, these suggest that the App NL-G-F/NL-G-F mice learnt and remembered the location similarly to their WT littermates.

App NL-G-F/NL-G-F mice show impairment in novelty-promoted memory.
Next, we investigated the effect of peri-encoding novelty in memory persistence. After one weak encoding trial with 2 pellets 21 , mice explored a box with novel substrates for 5-min or remained in the home cage. After a retention period of 24 or 6 h after encoding, a probe test was conducted to assess LTM or ITM (Fig. 1g). Weak encoding alone was insufficient to induce LTM as both genotypes performed indifferent from chance ( Fig. 1i  Novelty through exploration of novel objects after encoding was introduced (Fig. 1h). The results showed that encoding followed by novel object exploration, compared to encoding alone, led to ITM in the WT mice (Fig. 1k, t 11 = 2.69, p = 0.021). The genotype effect was significant (Fig. 1k, F 1,23 = 4.37, p = 0.048) although the interaction only trended towards significance (F 1 , 23 = 3.02, p = 0.095). A replication of this procedure with a different set of novel objects further supported this genotype difference (Fig. 1l, genotype: t 23 = 2.76, p = 0.011). Together, these results would suggest an impairment of peri-learning novelty in promoting memory in the AD mice.
Ageing effect in mice in the ADMP task. It has been reported that rats can retain their training performance from adulthood to middle-age with a long gap of no training 23 . Hence, we asked whether mice could maintain their performance over time (Fig. 2a). At 12-month-old, errors did not decrease over training blocks (Fig. 2b, F 4,92 = 0.94, p = 0.4). The genotype effect and interaction were insignificant (F 1,23 = 1.10, p = 0.3; F 4,92 = 1.03, p = 0.4, respectively). Critically, errors in the first block were not significantly below chance (Fig. 2b, t 24 = − 1.39, p = 0.2). This would suggest that the error-based performance did not maintain from adulthood to middle age.
At middle age, the latency to retrieve the reward reduced across training blocks (Fig. 2c, F 3,64 = 4.60, p = 0.007; a significant linear trend, F 1,23 = 12.39, p = 0.002). Genotype and interaction effects were insignificant (F 1,23 = 0.13, p = 0.7; F 3,65 = 0.76, p = 0.5, respectively). Critically, the latency at the first block at 12-month-old was significantly higher than the latency at the last block at adulthood (Paired samples Wilcoxon Signed Ranks p < 0.001). This would suggest that the task efficiency does not maintain to an older age although it improves after further training. While there was not a significant genotype effect in efficiency overall, AD mice were less efficient in the last training block (Fig. 2c, block 5, Mann-Whitney U = 29, p = 0.007).

Intact memory in the Barnes maze in App NL-G-F/NL-G-F
mice, although they take longer to reach the target. The Barnes maze task involved the use of aversive stimuli to motivate animals to locate an escape box. Learning was demonstrated by decreasing latencies to reach the escape box across training sessions (Fig. 3a,b, training session: F 2.4,55 = 5.29, p = 0.005). The interaction was insignificant (F 2.4,55 = 0.83, p = 0.5), which suggests that both groups acquired the task at a similar rate. However, App NL-G-F/NL-G-F mice took significantly longer to reach the escape box (F 1,23 = 6.46, p = 0.018). This was not due to genotypic differences in motor ability as speed did not differ between the two genotypes (F 1,23 < 0.1, p = 1) and their speed increased over training sessions (F 4,92 = 3.78, p = 0.007) with no significant group by session interaction (F 4,92 = 1.60, p = 0.2).
In the STM probe, there was no significant genotype effect (Fig. 3c, F 1,23 = 1.73, p = 0.2). The time spent in the target quadrant was significantly higher than in the other quadrants averaged (F 1,23 = 17.14, p < 0.001). The genotype-quadrant interaction was not significant (F 1,23 = 1.74, p = 0.2). In the LTM probe, there was also no significant genotype difference (Fig. 3d, F 1,23 = 0.287, p = 0.597). The time spent in the target quadrant was also significantly higher than in the other quadrants averaged (F 1,23 = 31.23, p < 0.001). The genotype-quadrant interaction was not significant (F 1,23 = 0.285, p = 0.599). Thus, the Barnes maze task did not reveal a robust genotype effect on STM or LTM. For novel object recognition, we asked if the animals learned and remembered the task at the test in contrast to the performance in sampling (Fig. 4d), if there was a group difference based on the genotype, and if aging from adulthood to the middle age affects recognition. To answer these, we performed a 3-way ANOVA on the data. The animals showed obvious learning and remembering, which effect was supported by a significant increase in the discrimination index from sampling to testing (Fig. 4e, F 1,23 = 85.83, p < 0.0001). The genotype effect was not significant (F 1,23 = 1.8, p = 0.2). The aging effect (F 1,23 = 0.04, p = 0.8) was not significant. All 2-way and 3-way interactions were not significant (all F 1,23 = 0.002-1.91, all p = 0.2-1.0). We verified that the performance at the baseline discrimination at sampling was not significantly different from chance (Fig. 4e, t tests against zero, WT: p = 0.7, App NL-G-F/NL-G-F : p = 0.2) and the performance at testing was significantly above chance (t tests against zero, both groups with p < 0.001). These results would suggest that the performance in the NOR task and the degree of learning in the App NL-G-F/NL-G-F mice remain comparable with those in the WT mice.

Intact open field exploration, spontaneous alternation, and recognition memory in
Similar analytical approaches were applied to data from the novel object location task (Fig. 4f). The animals showed obvious learning and remembering, which effect was supported by a significant increase in the discrimination index from sampling to testing (Fig. 4g, F 1,23 = 23.87, p < 0.0001). The genotype effect was not significant (F 1,23 = 2.86, p = 0.1). The aging effect (F 1,23 = 0.5, p = 0.5) was not significant. All 2-way and 3-way interactions were not significant (all F 1,23 = 0.02-0.61, all p = 0.4-0.9). We verified that the performance at the baseline discrimination at sampling was not significantly different from chance (Fig. 4g, t tests against zero, WT: p = 0.4, App NL-G-F/NL-G-F : p = 0.4) and the performance at testing was significantly above chance (t tests against zero, both p < 0.01). These results would suggest that the performance in the NOL task and the degree of learning in the App NL-G-F/NL-G-F mice remain comparable with those in the WT mice.
Amyloid load at middle age. Since genotype difference in memory impairment was mainly seen in behavioral tagging but not other measurements even at middle age, we set out to determine if the plaque burden in the hippocampus was similar to what had been previously reported. At the end point around 14 months old, App NL-G-F/NL-G-F mice showed clear amyloid pathology. In the hippocampus, the area covered in amyloid plaques was 1.8 ± 0.2% (Fig. 5a,b), which was significantly higher than 0 (t 11 = 7.98, p < 0.0001), but significantly lower than previously reported (t 14 = − 10.51, p < 0.0001, vs. 6.4% in 9-months old App NL-G-F/NL-G-F mice in Saito et al. 3 ). The number of plaques in the hippocampus was also significantly higher than 0 (Fig. 5c, 42.0 ± 4.4, t 11 = 9.56, p < 0.0001). The latencies were significantly reduced over 5 training days. The genotype effect, but not the interaction, was significant. (c) STM in both groups was indicated by significantly more time spent in the target quadrant than averagely in other quadrants (p < 0.001) and this was not affected by the genotype. (d) LTM in both groups was indicated by significantly more time spent in the target quadrant than averagely in other quadrants (p < 0.001) and this was not affected by the genotype. All data presented as mean ± SEM. *p < 0.05, ***p < 0.001. www.nature.com/scientificreports/ A schematic of the NOR task. (e) Learning of NOR led to significant increase in discrimination index from training to testing (p < 0.0001), while the genotype effect, aging effect, and interactions were not significant. (f) A schematic of the NOL task. (g) Learning of NOL recognition led to significant increase in discrimination index from training to testing (p < 0.0001), while the genotype effect, aging effect, and interactions were not significant.

Scientific Reports
(h) A schematic of the Y-maze. (i) Spontaneous alternation in the Y-maze was not affected by genotype. It was significantly above chance in both groups and at both ages (all p < 0.001). All data presented as mean ± SEM. ***p < 0.001.

Discussion
In this study, adult App NL-G-F/NL-G-F mice show impaired novelty-promoted memory (via BTC) in the ADMP task while STM and LTM in this task, STM and LTM in the Barnes maze, LTM in NOR, and ITM in NOL remain intact (Table 1). This ADMP task also sensitively reveals age-dependent decline in LTM in early aging. The intact LTM in adult App NL-G-F/NL-G-F mice may suggest that strong encoding would lead to sufficient tagging and PRPs in the activated cell populations in the hippocampus to sustain plasticity 29,41 or structure changes 42-44 for memory, despite amyloid pathology in the hippocampus at this stage 3,10,45 . Peri-encoding novelty facilitates memory persistence in WT mice, but not in App NL-G-F/NL-G-F mice. Using an overexpression model, APP/PS1 mice show weaker inhibitory avoidance learning compared to WT and impaired memory association through a BTC paradigm 46 . Here, we are able to rule out confounding from learning as App NL-G-F/NL-G-F mice show comparable learning abilities with WT mice in this ADMP task and the findings suggest that the impairment in novelty-promoted memory precedes apparent learning or memory decline. It has been proposed that BTC relies on synaptic meta-plasticity processes and that memories persist when learning events set tags and events providing PRPs occur in spatial and in temporal proximity 25 . One underlying mechanism in causing the impairment in novelty-promoted memory in AD mice could be due to a decrease in the PRP synthesis.
Novelty-promoted memory in WT animals in this study (Fig. 1k,l) and in an earlier aging study (see Fig. 3E in Gros and Wang 23 ) refers to an intermediate delay between encoding and testing. The intermediate-term memory has been shown to require protein synthesis 47,48 , although this has not been explicitly tested in this study. Whether object novelty promotes memory persistence at a longer delay (e.g. 24 h) in mice requires future investigation. The term 'memory persistence' can refer to LTM with a 24-h delay in the context of characterising the underlying receptor or molecular mechanisms on memory consolidation 49,50 . For example, it has been shown that protein synthesis is required for LTM, but not STM 51,52 , and for post-reactivation LTM but not postreactivation STM 53,54 . Novelty is often introduced within a short time window around encoding or reactivation to promote memory persistence 20,22,30 . When looking at a longer-term memory at 7 days, Tomaiuolo et al. show that exploration in a novel open field at 11 h, but not 5, 8, or 24 h after learning can promote 7-day memory in inhibitory avoidance 55 . Whether this type of delayed novelty can promote longer-term memory in the ADMP task in a reactivation-independent manner 55 or in a reactivation-dependent manner 22,56 requires future research.
Novelty modulates neuronal activities in the locus coeruleus and ventral tegmental area, which send dopaminergic projections to the hippocampus 21 . Blocking dopaminergic receptors in the hippocampus prevents peri-encoding novelty from facilitating memory persistence 20,30 . Critically, blocking dopaminergic receptors in the hippocampus also prevents locus coeruleus stimulation from facilitating memory persistence 21 . Collective evidence leads to the view that locus coeruleus and ventral tegmental area send projections to the hippocampus which enables novelty detection 46,57 and that such cross-region projections enable novelty-facilitated memory persistence through BTC 21,58 . Amyloid pathology is apparent in these midbrain structures in AD mouse models 59,60 and in App NL-G-F/NL-G-F mice 10,61,62 . Loss of dopaminergic neurons in midbrain is seen in familial AD mouse models with alpha-synuclein pathology 63 or presenilin mutations 60 . Reduced locus coeruleus volumes are reported in people with mild cognitive impairment or dementia due to AD 64 and brainstem volume reduction and deformation in people with clinical AD 65,66 . Together, it is likely that the circuits between midbrain and hippocampus are already affected at a younger age of AD mice 62 which contribute to impaired novelty-promoted memory (via BTC). www.nature.com/scientificreports/ At the cellular level, we have recently shown that encoding of the ADMP task and novelty engage overlapping cell populations in CA1 and CA3 of the hippocampus 24 . Further analyses reveal that novelty, compared to familiarity, preferentially activates more cells in the distal CA1 and increases the overlapping cell population in distal CA1 and proximal CA3 24 . The proximal CA3-distal CA1 regions and connection have been implicated in object novelty or recognition 67,68 . It is possible that exposure to novel objects activates this network more so than exploring a novel context or substrates. Longer exposure to a novel context or substrates beyond what we did in this study may be required to facilitate memory persistence in mice 32,69,70 . As peri-encoding novelty does not improve memory persistence in App NL-G-F/NL-G-F mice, future investigation in the local CA3-CA1 circuit can verify if AD causes alterations of this circuit and leads to impairment in novelty-promoted memory 71,72 . Disruptions to glutamate transmission at the CA3-CA1 synapses have been observed following pathology onset in the App NL-G-F/NL-G-F mice 73 . Indirect evidence hints toward distal, compared to proximal, CA1 being more vulnerable in showing pathology in early AD 74 . www.nature.com/scientificreports/ Aging-dependent decline in LTM is sensitively revealed with the ADMP task. This impairment in middle age is consistent with what has been shown in a cross-sectional study 24 and in a longitudinal study 23 in rats, both suggesting an encoding impairment in early aging. While encoding would also be affected by aging in the App NL-G-F/NL-G-F mice, the impairment in novelty-promoted memory at a younger age would suggest that early amyloid pathology may already affect production of PRP 29,75 and/or the circuits mentioned above for novelty detection in contributing to PRP.
Our open field results are consistent with previous studies that show comparable performance in wildtype and App NL-G-F/NL-G-F mice 7,10 , which suggests that App NL-G-F/NL-G-F mice are not more anxious. While both STM and LTM are comparable between groups in the Barnes maze task, App NL-G-F/NL-G-F mice take longer to reach the target. As they improve across training sessions, this does not reflect a learning deficit. The walking speed is also comparable between groups, suggesting no motor impairment. One potential explanation is that they are less anxious than wildtype mice in an environment with stronger aversive stimuli. Consistently, App NL-G-F/NL-G-F mice spend more time in the open arms in an elevated plus maze, suggesting reduced anxiety in this model 4 . Whether an anxiogenic or anxiolytic profile is observed may depend on the age of the animal, the progress of pathology, the behavioral history of the animals, and the magnitude of anxiogenic stimuli 76 .
Consistent with previous NOR studies, App NL-G-F/NL-G-F mice performed similar to wildtype mice 7,10 . Similar to NOR, NOL in our study fails to serve as a sensitive behavioral marker for AD phenotypes in App NL-G-F/NL-G-F mice. Our results of spontaneous alternation in the Y-maze are in agreement with an earlier study 7 . While another study showed poorer alternation in the App NL-G-F/NL-G-F mice, it is important to note that those App NL-G-F/NL-G-F mice still performed significantly above chance 3 . The subtle differences across studies could reflect environmental factors, such as experimenters' gender and cage enrichment which can affect animal stress levels 7,77,78 .
Notably, by middle-age our App NL-G-F/NL-G-F mice had significantly lower hippocampal amyloid plaque load than reported at 9 months 3 . The longitudinal design of the study with repeated tasks may have had a protective effect on the development of the pathology as repeated spatial training 79 , treadmill running 80 and environmental enrichment 81 have all been shown to reduce amyloid levels in AD models.

Conclusion
The App NL-G-F/NL-G-F mouse model provides a new opportunity to revisit the effects of amyloid on cognition without APP overexpression. The results show impairment in novelty-promoted memory (via BTC) prior to significant deficits in learning or memory in these mice. The circuit mechanisms, such as midbrain-hippocampal projections and CA3-CA1 circuits, underlying this type of early impairment with amyloid pathology requires future investigation. In conjunction with other studies highlighting synaptic tagging and capture impairments before apparent cognitive decline 75,82 , this could provide a new diagnostic direction for early detection of ADrelated functional impairment.

Data availability
The datasets generated and analysed during the current study are available from the corresponding author on request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.